S100A9 belongs to the S100-family of calcium-binding proteins and has been recognized as an attractive novel therapeutic target for the treatment of e.g. autoimmunity, inflammatory disease, neurodegenerative disease and cancer. Other S100 proteins have distinct roles in many different biological processes and are connected to a number of diseases including cancer, cardiomyopathies, atherosclerosis, Alzheimer's disease and inflammatory diseases. Twenty-one of the human genes, including S100A9, are located at chromosomal region 1q21, which is frequently altered in tumors (Marenholz et al., 2004). Interestingly, although the primary sequence diverges between family members, the 3D-structures of the different proteins are very similar.
S100A9 is often co-expressed with S100A8, another member of the S100 protein family, and they are highly expressed in myeloid cells, such as neutrophils and monocytes, but can also be induced in other cells or tissues (Srikrishna 2012). They form non-covalent homo- and heterocomplexes that can be specifically released in response to cellular activation (Foell et al., 2007, Ryckman et al., 2003). S100A9 can functionally be described as a damage-associated molecular pattern (DAMP) molecule which is released in tissues and induces signaling by interacting with receptors such as RAGE and TLR4 (Foell et al., 2007). As for many other DAMP molecules, S100A9 also has intracellular roles in addition to its extracellular functions, e.g. by binding to the cytoskeleton and influencing cytoskeletal rearrangements and thereby cellular migration (Srikrishna 2012). A pro-inflammatory role for S100A9 is supported by elevated S100A9 serum levels in inflammatory diseases and by high concentrations of S100A9 at local sites of inflammation, for example in the synovial fluid of rheumatoid arthritis patients (Foell et al., 2004) or osteoarthritis patients (van Lent 2012) where high levels correlate with joint destruction. Also, preclinical studies with S100A9 knock-out mice show an involvement of S100A9 in many inflammatory processes including synovial activation and cartilage destruction during osteoarthritis (van Lent 2012). High levels of S100A9 have also been found in several forms of cancer and a high expression level has been shown to correlate with poor tumor differentiation in some of these cancer forms (Arai et al., 2008). Elevated S100A9 levels in pathological conditions of chronic inflammation as well as in cancer argue for a possible role in inflammation-associated carcinogenesis.
A role for S100A9 in the coupling between the immune system and cancer is also supported by studies showing that S100A8 and S100A9 are highly expressed in and important for the function of myeloid-derived suppressor cells (MDSCs) (Cheng et al., 2008, Sinha et al., 2008, Wang et al., 2013), a mixture of immature myeloid cells that suppress T- and NK-cell activation and promote angiogenesis and tumor growth. By interfering with S100A9-regulated accumulation of tumor infiltrating MDSCs, the balance between these processes may change in favor of an anti-angiogenic and less immune suppressive milieu with inhibited tumor progression. Furthermore, there are data suggesting a role for S100A9 in recruiting both inflammatory cells and tumor cells to metastatic sites (Hiratsuka et al., 2006, Acharyya et al. 2012, Hibino et al., 2013). Thus, blocking the function of S100A9 may provide a new approach to prevention of metastasis.
Although a number of possible biological functions of S100A9 have been proposed, the exact role of S100A9 in inflammation, in cancer and in other diseases is still unknown. Members of the S100 protein family have been reported to interact with the pro-inflammatory molecule RAGE and studies showed that S100A9 is the strongest RAGE binder within the S100 family in the presence of physiological levels of Ca2+ and Zn2+ (Bjirk et al. 2009). These studies further demonstrated that S100A9 interacts with toll-like receptor 4 (TLR4). As for the S100A9-RAGE interaction, the S100A9-TLR4 interaction appears to be strictly dependent on the presence of physiological levels of both Ca2+ and Zn2+. Another receptor for S100A9 that may be important in cancer is EMMPRIN (CD 147), this protein is expressed on different cell types and the S100A9-EMMPRIN interaction has been shown to be involved in melanoma metastasis (Hibino et al., 2013).
S100A8 and S100A9 proteins have predominantly been described as cytoplasmic proteins that are secreted from myeloid cells upon activation. It is generally believed that the major biological functions relevant to inflammation require the release of the S100 proteins to the extracellular space. In this model, extracellular S100A9 would bind to e.g. the pro-inflammatory receptors RAGE and TLR4 and result in an inflammatory response. This is supported by studies showing that S100A9 induces TNF production in human monocytes via TLR4 (Riva et al. 2012, Cesaro et al. 2012). Also, S100A9 in complex with S100A8 has shown growth promoting activity directly on tumors cells via RAGE signaling (Ghavami et al., 2008). S100A9 also exists in a membrane-associated form on monocytes (Bhardwaj et al., 1992). Membrane associated S100A9 opens up for the possibility of cell-cell or cell-ECM signaling involving S100A9.
The collected data suggest that S100A9 have important roles in inflammation, cancer growth, cancer metastasis and in their connections. Novel compounds that inhibit the activity of S100A9 in these processes, and thereby disturb the tumor microenvironment, would be attractive in treatment of cancer of different types.
Besides cancer, inflammation and autoimmunity, S100A9 has strong connections to neurodegenerative disease. S100A9 is upregulated in the brain in Alzheimer's disease (AD) patients and in mouse disease models (Shepherd et al., 2006, Ha et al., 2010). Furthermore, knock-down or deletion of S100A9 in mice models of AD inhibits cognition decline and plaque burden in the brain (Ha et al., 2010, Chang et al., 2012). A role for RAGE is also evident in AD where inhibition of RAGE reduces disease in a mouse AD model (Deane et al., 2013). Inhibition of S100A9 and its interactions represents a new promising approach for therapeutic intervention in AD and other neurodegenerative diseases.
WO 02/069965 (Transtech Pharma Inc) discloses certain benzimidazole derivatives, as modulators of the interaction between RAGE and its ligands for the management, treatment, control, or as an adjunct treatment for diseases in humans caused by RAGE, e.g. acute and chronic inflammation, the development of diabetic late complications such as increased vascular permeability, nephropathy, atherosclerosis, and retinopathy, the development of Alzheimer's disease, erectile dysfunction, and tumor invasion and metastasis.
A number of publications describe pharmacological effects (often low to moderate) of imidazo[2,1-b]thiazole derivatives, viz. 7-thia-2,5-diazatricyclo[6.4.0.02,6]dodeca-1(8),3,5,9,11-pentaene-4-carboxylic acids and imidazo[2,1-b][1,3]thiazole-6-carboxylic acids. Thus, analgesic and antiinflammotory effects were described by Palagiano 1995, Palagiano 1996, Abignente 1981 and Grandolini 1993. Antiallergic effect was reported by Ager 1988, anxiolytic effect by Clements-Jewery 1988 (inactive in flunitrazepam receptor binding) and SIRT1 activation by Vu et al 2009 (inactive). In addition, blood sugar reducing effect was reported in U.S. Pat. No. 4,137,320 and activity against hepatitis C was reported in WO2006008556. Other publications describe 7-thia-2,5-diazatricyclo[6.4.0.02,6]dodeca-1(8),3,5,9,11-pentaene-4-carboxylic acids and imidazo[2,1-b][1,3]thiazole-6-carboxylic derivatives as synthesis targets or synthetic intermediates with no pharmacological data e.g. Blackburn 2010, Herath 2010 and Patel 2009.
A number of imidazo[2,1-b]thiazole derivatives are commercially available, or have been disclosed in the literature, but have not hitherto been disclosed for use in therapy.